EXO84B Antibody

Basic Research Questions

• What is the optimal dilution for EXO84B Antibody in Western blotting?

For Western blotting applications, the optimal dilution range for EXO84B Antibody is typically 1:1000-1:6000 in 5% BSA in TBST. This should be empirically determined for each application as it depends on protein expression levels and detection system sensitivity. Begin with 1:1000 dilution for standard cell lysates (20-30μg total protein) and adjust as needed. Overnight incubation at 4°C generally yields optimal signal-to-noise ratio compared to shorter room temperature incubations.

Western blot optimization requires careful consideration of several experimental variables:

• Sample preparation: Lyse cells in RIPA buffer containing protease inhibitors to prevent protein degradation

• Loading controls: Include β-actin or other housekeeping proteins to normalize for loading differences

• Blocking buffer: 5% non-fat dry milk or BSA in TBST, optimized for your specific antibody

• Secondary antibody selection: For mouse monoclonal primary antibodies like EXO84B (H-1), use anti-mouse IgG conjugated to HRP or fluorophores

• Detection system: ECL substrates of appropriate sensitivity for chemiluminescent detection, or appropriate imaging systems for fluorescent detection

Working dilutions should be prepared fresh and not stored for extended periods, as antibody activity can diminish over time.

• What is the recommended protocol for immunofluorescence using EXO84B Antibody?

For immunofluorescence applications with EXO84B Antibody:

• Fix cells with 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 in PBS (10 minutes)

• Block with 5% normal serum in PBS (1 hour)

• Incubate with primary EXO84B Antibody at 1:50-1:500 dilution (overnight at 4°C)

• Wash 3× with PBS (5 minutes each)

• Incubate with fluorophore-conjugated secondary antibody (1-2 hours at room temperature)

• Counterstain nuclei with DAPI (1:1000 dilution, 5 minutes)

• Mount and image

EXO84B typically shows punctate cytoplasmic staining with enrichment at membrane exocytosis sites and the midbody during cytokinesis. When troubleshooting immunofluorescence experiments, consider these critical factors:

• Fixation method and duration significantly impact epitope accessibility

• Detergent concentration must be optimized to balance membrane permeabilization with preservation of cellular structures

• Blocking with appropriate serum (matched to secondary antibody species) reduces non-specific binding

• Signal amplification systems may be necessary for low-abundance targets

• Negative controls (primary antibody omission, isotype controls) should be included to verify specificity

For co-localization studies, combine EXO84B antibody with markers for cellular compartments such as ER (CFP:HDEL), plasma membrane, or other exocyst components .

• What storage conditions are required for EXO84B Antibody to maintain activity?

EXO84B Antibody requires specific storage conditions to maintain optimal activity:

• Store at -20°C in a non-frost-free freezer

• Store in aliquots (10-50μl) to prevent freeze-thaw cycles

• Add glycerol to 50% final concentration for long-term storage

• Sodium azide (0.02%) may be added as a preservative, but note it can inhibit HRP in subsequent applications

• Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• For short-term use (1-2 weeks), store at 4°C

• Working dilutions should be prepared fresh and not stored for extended periods

Properly stored, the antibody typically maintains activity for 12-24 months from receipt date. The molecular stability of antibodies is affected by multiple factors that researchers should consider:

• Physical agitation can lead to denaturation and aggregation

• Oxidation and light exposure accelerate antibody degradation

• Stabilizers like BSA (0.1%) can improve long-term stability

• Concentration changes from evaporation during repeated freeze-thaw cycles affect performance

• Microbial contamination can occur without proper preservatives

For antibody validation before critical experiments, perform Western blot analysis using positive control lysates from cell lines with known EXO84B expression to confirm antibody activity and specificity .

Advanced Research Questions

• How can EXO84B phosphorylation be detected and what are its functional implications?

Detection of EXO84B phosphorylation requires specialized techniques:

• Phospho-specific antibodies: When available, these directly detect specific phosphorylated residues

• Phos-tag™ SDS-PAGE: Incorporates Phos-tag™ molecule that specifically retards phosphorylated proteins, creating mobility shifts

• λ-phosphatase treatment: Compare migration patterns before/after treatment

• Mass spectrometry: For precise identification of phosphorylation sites

Functional implications include:

• Disruption of exocyst complex assembly (phosphorylation at Ser743 by CDK1)

• Regulation of vesicle tethering during cell cycle progression

• Modulation of interaction with Ral GTPases

• Control of exocyst localization during cytokinesis

Phosphorylation typically reduces EXO84B binding to other exocyst components by 60-80% as measured by co-immunoprecipitation assays. Research has demonstrated that CDK1-mediated phosphorylation of EXO84B significantly impacts its interaction with other exocyst subunits:

Experimental validation using phosphomimetic (S→D/E) and phospho-null (S→A) mutations has confirmed these modifications function as molecular switches regulating exocyst assembly and disassembly during various cellular processes .

• What bimolecular fluorescence complementation (BiFC) strategies can be used to study EXO84B interactions?

BiFC strategies for studying EXO84B interactions require careful experimental design:

• Construct design: Fuse N-terminal (VN) and C-terminal (VC) fragments of Venus/YFP to EXO84B and potential interaction partners

  • Critical consideration: fusion orientation affects complex formation; both N- and C-terminal fusions should be tested

  • Domain-specific tagging: Consider EXO84B's domain structure (N-terminal coiled-coil, C-terminal PH domain)

• Controls:

  • Positive: Known interaction partners (e.g., SEC8, SEC10)

  • Negative: Non-interacting proteins with similar localization

  • Expression level controls: Western blots to verify comparable expression

• Analysis protocols:

  • Live-cell imaging at 37°C in phenol red-free media

  • Fixed time-point analysis (24-48h post-transfection optimal)

  • Quantification of BiFC signal intensity and localization

• Advanced applications:

  • Multicolor BiFC to visualize multiple interactions simultaneously

  • BiFC combined with FRET to analyze complex formation dynamics

  • Photo-activatable BiFC for temporal control of visualization

When implementing BiFC for EXO84B interaction studies, researchers should be aware of certain methodological considerations:

• BiFC complex formation occurs with a delay due to the slow rate of fluorophore maturation (typically 50 min half-time)

• Some BiFC complexes form irreversibly, making them unsuitable for dynamic interaction studies

• The YN155 (residues 1-154) and YC155 (residues 155-238) fragments of YFP often provide optimal signal-to-noise ratio

• Venus fragments may produce brighter fluorescence but also higher background

• Efficiency of fluorescence complementation is typically <10% of intact fluorescent protein expression

The BiFC approach has successfully revealed specific interactions between EXO84C and VAP27-1/3 in plant cells, demonstrating the utility of this technique for studying exocyst component interactions in various biological systems .

• How does EXO84B contribute to exocyst complex architecture based on structural studies?

EXO84B makes critical contributions to exocyst complex architecture:

• Structural organization: Cryo-EM studies at 4.4Å resolution reveal EXO84B forms an extended rod-like structure composed primarily of α-helical bundles

• Domain interactions:

  • N-terminal region (aa 1-200): Forms coiled-coil interactions with SEC10

  • Central region (aa 201-550): Provides structural scaffold

  • C-terminal region (aa 551-784): Contains PH domain that binds phosphoinositides

• Assembly interfaces:

  • Primary: EXO84B-SEC10 interface (buried surface area ~2600Ų)

  • Secondary: EXO84B-SEC15 interface (buried surface area ~1200Ų)

• Subcomplex formation: EXO84B participates in a subcomplex with SEC10, SEC15, and EXO70 that can exist independently of the full octameric complex

• Conformational states: EXO84B undergoes significant conformational changes upon complex assembly, with a ~30° rotation of its C-terminal domain

Recent cryo-electron tomography studies have revealed two potential conformational states of the exocyst during membrane tethering:

• The exocyst positioned between secretory vesicles and plasma membrane, yielding 32nm membrane separation

• The exocyst positioned aside to the vesicle-plasma membrane contact interface, yielding minimal membrane separation

Chemical cross-linking mass spectrometry (CXMS) has further elucidated the interaction network within the exocyst, confirming that EXO84B and EXO70 form a distinct pair within the complex through intertwined coiled-coil interactions termed "CorEx" (Core of Exocyst) .

• What are the comparative properties of EXO84B across different species?

EXO84B properties vary across species, with implications for experimental design and comparative studies:

Domain abbreviations: CC = Coiled-coil, PH = Pleckstrin Homology

In plants, multiple EXO84 isoforms (EXO84a, EXO84b, EXO84c) show distinct interaction patterns and subcellular localizations. For example, EXO84c specifically interacts with VAP27-1/3 proteins at ER-derived structures, while EXO84a and EXO84b do not show this interaction . This functional diversification suggests specialized roles for EXO84 paralogs in different organisms.

Antibody cross-reactivity studies have demonstrated that the H-1 monoclonal antibody against human EXO84B has high specificity for mammalian orthologues (human, mouse, rat) but may not recognize more divergent forms in other species . When conducting comparative studies, researchers should verify antibody specificity for each species of interest.

• What effect does EXO84B phosphorylation have on exocyst complex assembly?

EXO84B phosphorylation significantly impacts exocyst complex assembly in a site-specific manner:

Experimental validation using phosphomimetic (S→D/E) and phospho-null (S→A) mutations demonstrates that these modifications serve as molecular switches regulating exocyst assembly and disassembly during various cellular processes. This dynamic regulation is particularly important during cell cycle progression and in response to extracellular signals .

Molecular mechanism studies have revealed that phosphorylation of EXO84B at Ser743 by CDK1 specifically disrupts its direct interaction with SEC10. In yeast models, mutation of the five phosphorylation sites of Exo84 to alanine ("Exo84-A") enhances interactions with Sec10, Sec15, and Exo70, while phosphomimetic mutations ("Exo84-E") diminish these interactions .

The functional consequences of EXO84B phosphorylation have been demonstrated through secretion assays and electron microscopy:

• Cells expressing phosphomimetic Exo84-E mutants show:

  • Accumulation of secretory cargo (e.g., Bgl2 in yeast)

  • Increased number of 80-100nm secretory vesicles visualized by EM

  • Enhanced secretion defects when combined with other exocyst mutations

• Cells expressing phospho-deficient Exo84-A mutants show:

  • Normal or enhanced secretion efficiency

  • Rescue of secretion defects in exocyst mutant backgrounds

  • Fewer accumulated secretory vesicles